Cell-image analyzing apparatus

ABSTRACT

A cell-image analyzing apparatus is intended to analyze, using a cell image, a cell collective that forms a colony, and is provided with a computer. The cell-image analyzing apparatus has an image analysis software that makes the computer function as a boundary element extracting means for extracting boundary elements of subjects upon analyzing the cell image; a possible colony region determining means for determining, as a possible colony region, a region surrounded by boundary elements of subjects and having a size greater than a first criterion value; and a colony region determining means for determining, in the possible colony region, a region containing more than a predetermined number of clustered regions each being surrounded by boundary elements of subjects and having a size smaller than a second criterion value, as a colony region.

This application claims benefits of Japanese Patent Application No.2009-1.73473 filed in Japan on Jul. 24, 2009, the contents of which arehereby incorporated by reference.

BACKGROUND OF THE INVENTION

1) Field of the Invention

The present invention relates to a cell-image analyzing apparatus forautomatically analyzing a collective of clustered cells (i.e. cellcolony) using cell images.

2) Description of the Related Art

Analysis of a cell colony is conducted normally for observing cells ascultured. As an apparatus for conducting analysis of a cell colony,there is conventional one, for example, referred to in the operationmanual “Analysis Software Operation, CELAVIEW RS100”, ver. 1.4, pp.3-17, published by Olympus Corporation.

When special cells such as stem cells or ordinary cancer cells arecultured, the cells divide and grow. In such a situation, cells oftencluster to form a colony. Therefore, automatic analyses of colonies arevery frequently needed.

Cell analyses intended for colonies as subjects include various typessuch as those to determine whether cultured cells form a colony or not,dimensions of a colony, growth speed of a colony, etc. Upon analyzingsuch information on colonies, a screening, for example, of agents thatpromote or inhibit growth is made. Since a large number of samples andcell images are required for screening of agents in either cellanalysis, it is necessary to automatically conduct the analysis ofcolonies.

A problem peculiar to colony analysis is in that, in a situation where acell collective that forms a colony and cells other than those existclose to each other, it is necessary to distinguish the cell collectivethat forms a colony from the remaining cells. For example, there aresome situations where some cells, out of cultured cells, form a colonyand the remaining cells independently exist without forming a colony.Regarding stem cells, in particular, immature or undifferentiated cellsform a colony while mature or differentiated cells independently existwithout forming a colony. In such a case, a cell collective that forms acolony should be distinguished from mature or differentiated cells whichindependently exist.

As another example, there is a situation where plural kinds of cellscoexist and only one kind of the cells form a colony.

As still another example, there is a situation where cells called“feeder cells” are simultaneously cultured with subject cells, forpromoting growth of the subject cells or keeping alive the subjectcells. In this case, it is necessary to distinguish the cell regionsforming a colony, as the subject, and the remaining cell regionsincluding the feeder cells.

SUMMARY OF THE INVENTION

A cell-image analyzing apparatus of the present invention is intended toanalyze, using a cell image, a cell collective that forms a colony, andis provided with a computer. The cell image analyzing apparatus has animage analysis software that makes the computer function as a boundaryelement extracting means for extracting boundary elements of subjectssuch as cells, intracellular organelles, and dust particles, uponanalyzing the cell image; a possible colony region determining means fordetermining, as a possible colony region, a region surrounded byboundary elements of subjects and has a size greater than a firstcriterion value; and a colony region determining means for determining,in the possible colony region determined via the possible colony regiondetermining means, a region that contains more than a predeterminednumber of clustered regions each being surrounded by boundary elementsof subjects and having a size smaller than a second criterion value, asa colony region.

In the cell-image analyzing apparatus of the present invention, it ispreferred that the boundary element extracting means extracts, asboundary elements of subjects, pixel portions where luminance greatlychanges and that the colony region determining means has the function ofdigitizing intricacy of boundary elements of subjects and of dividingthe possible colony region into a colony region and a non-colony regionin accordance with the intricacy of boundary elements as digitized.

In the cell-image analyzing apparatus of the present invention, it ispreferred that the colony region determining means sets, in the possiblecolony region determined via the possible colony region determiningmeans, evaluation regions having a uniform shape and size, detectsvalues of boundary elements of subjects encircled in the evaluationregions in terms of number of pixels, and gives the detected values asthe intricacy.

In the cell-image analyzing apparatus of the present invention, it ispreferred that the colony region determining means has the function ofsetting, as a non colony cell model region, in the possible colonyregion, a circle, an ellipse, a square or other figure with a certainsize, which is determined in reference to the minimum size of a celloutside a colony as a criterion, and of detecting, upon shifting theposition of the non-colony cell model region in the possible colonyregion, a region where the non-colony cell model region is allowed to beset without encircling any boundary element of subjects, as a region ofa non-colony cell.

In the cell-image analyzing apparatus of the present invention, it ispreferred that the colony region determining means further has thefunction of determining regions of individual cells other than thenon-colony cell by setting a largest circle that has a radius smallerthan the non-colony cell model region and that encircles no boundaryelement and repeating this step of setting a circle inside the possiblecolony region.

In the cell-image analyzing apparatus of the present invention, it ispreferred that the colony region determining means has the function ofpredicting that, upon growing a marked region from an arbitrary originon the boundary elements of subjects in the possible colony region byrepeating the process of incorporating adjacent points into the markedregion until the marked region contains a preset average number ofboundary pixels (average value (number of pixels) boundary elements) fora single cell, the marked region as containing the average number ofboundary pixels is a region occupied by a single cell inside thepossible colony region.

In the cell-image analyzing apparatus of the present invention, it ispreferred that the colony region determining means further has thefunction of determining, in the possible colony region, whether eachregion predicted as occupied by a single cell belongs to a colony regionor a non-colony region based on a morphologic characteristic.

In the cell-image analyzing apparatus of the present invention, it ispreferred that the morphologic characteristic is circularity.

According to the present invention, it is possible to provide acell-image analyzing apparatus that is capable of automaticallyanalyzing a cell image, to be specific, accurately determining, in aregion in which regions forming a colony and regions not forming acolony coexist, the regions forming a colony and then acquiring andoutputting information such as position and size of the regions forminga colony.

These and other features and advantages of the present invention willbecome apparent from the following detailed description of the preferredembodiment when taken in conjunction of the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, is a block diagram that shows the entire configuration of acell-image analyzing apparatus according to one embodiment of thepresent invention.

FIG. 2, is a flow chart that shows the entire procedure of cell imageanalysis, from photographing for a cell image through analysis of thecell image using the cell-image analyzing apparatus of this embodiment.

FIG. 3 is a flow chart that shows the operation procedure ending withdetermination of a colony region by analyzing a cell image, using thecell-image analyzing apparatus of this embodiment.

FIGS. 4A, 4B and 4C are explanatory diagrams for illustrating states ofextraction of boundary elements of subjects from cell images by analysisof the cell images using the cell-image analyzing apparatus of thisembodiment, where FIG. 4A shows a state where a large number of boundaryelements of subjects are extracted, FIG. 4B shows a state where a normalnumber of boundary elements of subjects are extracted, and FIG. 4C showsa state where a small number of boundary elements of subjects areextracted.

FIG. 5 is a schematic diagram that shows one example of individualregions delimited by using boundary elements of subject in a cell image.

FIGS. 6A, 6B, 6C and 6D are explanatory diagrams that show one exampleof the cell-image analysis procedure according to the second mode in thecell-image analyzing apparatus of this embodiment, where FIG. 6A showsthe original cell state (cell image), FIG. 6B shows the state ofextracting boundary elements of subjects from the cell image of FIG. 6A,FIG. 6C shows the state of detecting a non-colony cell region, uponsetting a non-colony cell model region in the image with the extractedboundary elements of FIG. 6B, and FIG. 6D shows the state of determiningregions of individual cells other than the non-colony cell, upon settinga circle which is a largest circle encircling no boundary element ofsubjects with a radius smaller than the non-colony cell model region, inthe image with the detected non-colony cell region of FIG. 6C.

FIGS. 7A and 7B are explanatory diagrams that show one example of thecell-image analysis procedure according to the third mode in thecell-image analyzing apparatus of this embodiment, where FIG. 7A showsthe state of evaluating intricacy of boundary elements of subjects uponsetting evaluation regions having a uniform shape and size in thepossible colony region, and FIG. 78 shows a series of regions evaluatedas high-density regions regarding boundary elements of subjects.

FIGS. 8A, 8B and 8C are explanatory diagrams that show one example ofthe cell-image analysis procedure according to the fourth mode in thecell-image analyzing apparatus of this embodiment, where FIG. 8A showsthe basic method of predicting a region occupied by a single cell in thepossible colony region by using boundary elements of subjects, FIG. 8Bschematically shows the state of predicting a region occupied by asingle cell in the possible colony region using boundary elements ofsubjects in the situation where boundary elements of subjects exist notonly on the circumference of the cell but also inside the cell, and FIG.8C schematically shows the state of predicting a region occupied by asingle cell in the possible colony region using boundary elements ofsubjects in the situation where a part of the boundary elements aremissing.

FIG. 9 is an explanatory diagram that schematically shows the process ofdetermining individual cells in a possible colony region that contains aplurality of clustered cells, by the cell-image analyzing apparatus ofthis embodiment using the fourth mode.

FIGS. 10A and 10B are explanatory diagrams that schematically show cellregions determined by the fourth mode in a possible colony region thatcontains clustered cells, in the cell-image analyzing apparatus of thisembodiment, where FIG. 10A schematically shows the state where cells areclustered, and FIG. 10B shows the individual cell regions determined bythe fourth mode.

FIGS. 11A and 11B are explanatory diagrams that illustrate themorphologic characteristic of individual single-cell regions determinedby the fourth mode, where FIG. 11A illustrates a single-cell regiondetermined in a colony region, and FIG. 118 illustrates a single-cellregion determined in a non-colony region.

DETAILED DESCRIPTION OP THE PREFERRED EMBODIMENTS

FIG. 1 is a block diagram that shows the entire configuration of acell-image analyzing apparatus according to one embodiment of thepresent invention. FIG. 2. is a flowchart that shows the entireprocedure of cell-image analysis, from photographing for a cell imagethrough analysis of the cell image using the cell-image analyzingapparatus of this embodiment. FIG. 3 is a flow chart that shows theoperation procedure ending with determination of a colony region byanalyzing a cell image, using the cell-image analyzing apparatus of thisembodiment.

The cell-image analyzing apparatus 1 of this embodiment is provided witha computer, and conducts analysis of a cell collective that, forms acolony, using a cell image captured via a cell-image photographingapparatus such as a microscopic photographing apparatus. The cell-imageanalyzing apparatus has an image analysis sot software that makes thecomputer function as a boundary element extracting means 1 a, a possiblecolony region determining means 1 b, and a colony region determiningmeans 1 c.

The boundary element extracting means 1 a extracts line segment elementsthat form boundaries of subjects such as cells, intracellularorganelles, and dust particles. The possible colony region determiningmeans 1 b determines, as a possible colony region, a region surroundedby boundary elements of subjects and has a size greater than a firstcriterion value. The colony region determining means 1 c determines, inthe possible colony region determined via the possible colony regiondetermining means 1 b, a region that contains more than a predeterminednumber of clustered regions each being surrounded by boundary elementsof subjects and having a size smaller than a second criterion value, asa colony region.

The image analysis software is configured to make the computer functionas an analysis result output means 1 d also for outputting result ofcell-image analysis.

An analysis of a cell image using the cell-image analyzing apparatus ofthis embodiment thus configured is made as follows.

One example of the entire procedure of cell image analysis fromphotographing for a cell in age through determination of a colony in thecell image via image analysis is shown in FIG. 2. The cell-imageanalysis procedure is completed by serially conducting an experiment(photographing) preparation step (Step S1), a photographing step (Step22), a cell image analysis step, (Step S3), and an analysis resultoutput step (Step S4). The cell-image analyzing apparatus of thisembodiment commences its operation at the cell-image analysis step.

Experiment (Photographing) Preparation Step (Step 1)

At the experiment preparation step, staining or culturing of cells ismade. Regarding the cell-image photography, there are known, as typicalones, techniques of acquiring a fluorescence image upon treatment suchas staining and techniques of acquiring a transmitted cell image withoutstaining using an appropriate transmitted illumination system. Of thesetechniques of acquiring a transmitted cell image, there are knownseveral techniques such as that in which a phase-contrast microscopicimage is acquired or a transmitted image via a tilted illuminationsystem is acquired.

The cell-image analyzing apparatus of this embodiment analyzes cellimages using cell-image analysis methods that are commonly applicable tocell images acquired through these techniques.

Staining

Whether or not cell staining should be made depends on purpose and/orcondition of the experiment. When cells are stained, they are killed orchanged from their proper state. Therefore, in general, in an experimentwhere cells are observed as living, staining is not made. On the otherhand, since the fluorescence photography allows a much distinct image tobe acquired in comparison with the transmitted illumination photography.Therefore, in order to perform automatic cell analysis, it is desirableto conduct the experiment upon fluorescence staining.

Photographing Step (Step S2)

Regarding photographing for a cell image, cells stained or cultured in aculture container are photographed with a microscopic photographingapparatus not shown, as in the same container or upon transferred into adifferent container optimized for observation. In many cases, the numberof colonies and the location of colonization are unknown at thebeginning of experimentation. Therefore, at least at the beginning ofexperimentation, photographing shots are taken for a plurality of targetpoints, to cover the entire region in the container. In anexperimentation system in which the experiment is repeated forobservation of cell growth, observation (photographing) is made for aneighborhood covering the position of the colony detected in the latestexperiment at its center.

Cell-Image Analysis Step (Step S3)

The cell-image analyzing apparatus of this embodiment automaticallyanalyzes a cell image captured after the experiment preparation step andthe photographing step (Step S1, Step S2) in accordance with theanalysis procedure shown in FIG. 3. To be specific, the analysis is madeby extracting boundary elements of subjects from the cell image (StepS31), then determining a possible colony region (Step S32), and thendetermining a colony region in the possible colony region (Step S33).The operation of these steps is described in detail below.

Extraction of Boundary Elements of Subjects (Step 31)

First, the boundary element extracting means 1 a extracts boundaryelements of subjects in a cell image. Here, the boundary elements ofsubjects are line segments that represent boundaries of characteristicsubjects such as cells, intracellular organelles and dust particles. Theboundary elements of subjects can be extracted using luminance andluminance difference within, the region of the cell image.

In general, a region where a cell exists and a region where no cellexists differ in image brightness (luminance characteristic)irrespective of whether the image is a transmitted image or afluorescence image. Using this principle, detection is made for a regionof picture elements such as pixels where luminance or luminancedifference greatly changes beyond a preset criterion value of variationof the luminance or luminance difference, and this pixel region isextracted as boundary elements.

States of extraction of boundary elements of subjects from cell imagesby analysis of the cell images using the cell-image analyzing apparatusof this embodiment are illustrated in FIGS. 4A-4C, FIG. 4A shows a statewhere a large number of boundary elements of subjects are extracted.FIG. 48 shows a state where a normal number of boundary elements ofsubjects are extracted, and FIG. 4C shows a state where a small numberof boundary elements of subject are extracted.

Determination of Possible Colony Region (Step S32)

Then, the possible colony region determining means 1 b conductsdetermination of a possible colony region using boundary elements ofsubjects. Since the analysis subject of the cell-image analyzingapparatus of this embodiment is a colony, a region that is too small tocontain a smallest unit subject of a colony is excluded from theanalysis subject even if it contains boundary elements of subjects.Whereby, a region that possibly includes a colony is determined as apossible colony region.

To be specific, the possible colony region determining means firstdelimits individual regions using the boundary elements of subjects.That is, a region surrounded by boundary elements of subjects along withits neighboring region is delimited as one region. Regarding theboundary elements of subjects used to delimit the individual regions, insome cases lines they form are partially broken where they fail to beextracted by the boundary element extracting means 1 b as boundaryelements of subjects, because the variation of luminance or luminancedifference is small and indistinct in some pixel regions. Regardingboundary elements that form partially broken lines, the possible colonyregion determining means 1 b recognizes boundary elements separated fromeach other at a distance smaller than a preset value as boundaryelements of an identical subject, and interpolates the space betweenthese boundary elements with certain line segment elements. Theconfiguration may be made so that this preset value is programmable by auser preceding the analysis procedure.

At this stage, in a cell image with extracted boundary elements ofsubjects, there are delimited individual regions, for example as shownin FIG. 5, of a dust particle, an independent cell, a cell collectivethat forms a colony, and a peripheral cell collective other than thecolony.

Then, the possible colony region determining means 1 b determines aregion that has a size greater than a predetermined value of aparameter, such as area, representing the size of regions, as a possiblecolony region. This value is preliminarily set, as a first criterionvalue, to be greater than an independent cell. At this stage, of theregions shown in FIG. 5, the regions of the independent cell and of thedust particle smaller than the independent cell are excluded.

Determination of Colony Region (Step S33)

Then, the colony region determining means is determines, in the possiblecolony region having a substantial size greater than the first criterionvalue as determined via the possible colony region determining means 1b, a region satisfying the following conditions (2) and (3) as a regionwhere cells are clustered to form a colony.

Conditions of Colony Detection

The cell-image analyzing apparatus of this embodiment is configured todetect a region that satisfies the following conditions (1)-(3) as acolony:

-   -   Condition (1) the region in concern has a substantial size        (greater than a first criterion value);    -   Condition (2) individual subjects (cells) inside the region in        concern are small (smaller than a second criterion value);    -   Condition (3) the number of these subjects (cells) is greater        than a predetermined number.

Processing Corresponding to Condition (1)

Condition (1) is a condition for determining a possible colony region asa region that possibly includes a colony. As described above, in theprocess of determining a possible colony region (Step S32), the possiblecolony region determining means 1 b determines a region satisfyingCondition (1) as a possible colony region. Therefore, small regions suchas dust particles and independent cells are excluded.

Processing Corresponding to Condition (2)

Condition (2) is a condition for excluding a region in which cells areclustered but the individual clustered cells are large. The phenomenonof cell clustering is not unique to colonies, but commonly presentsitself in cells in culture. In particular, the phenomenon that maturecells simply exist close together to be clustered is very commonly seen.

Therefore, in the cell-image analyzing apparatus of this embodiment, thecolony region determining means 1 c predicts the size (for example,area) of each of individual subjects (cells) inside a possible colonyregion determined via the possible colony region determining means 1 band compares it with the second criterion value for the purpose ofdistinguishing between a collective of mature cells and a colony.Depending on the experiment, conditions and experiment purposes, someexperimentation systems do not require such distinguishing. Therefore,in the cell-image analyzing apparatus of this embodiment, it ispreferred that whether the colony region determining process by thecolony region determining means 1 c is necessary or not is preliminarilyprogrammable by the user.

In the cell-image analyzing apparatus of this embodiment, it ispreferred that a plurality of processing modes such as the laterdescribed first to fourth modes are selectable by a user to be used fordistinguishing between cells in a colony region and cells in anon-colony region. The difficulty in extraction of boundary elements ofsubjects differs by experimentation system. Therefore, it is preferredthat the user can freely choose, for each analysis, an appropriateprocessing mode among a plurality of processing modes such as the firstto fourth modes described later.

Challenges in Determination of Colony Region C-1—Difficulty inRecognition of Each of Clustered Cells

In determination of a colony region, if individual cells can berecognized upon determination of boundary elements of the individualsubjects in the possible colony region, it is easy to determinesatisfaction/non-satisfaction of Conditions (1)-(3) However, since cellsusually are clustered with an intricate pattern in a colony region,there is a problem that recognition of the individual cells isinherently difficult.

C-2—Challenges Derived from Difficulty in Extraction of BoundaryElements of Subjects

Also, since accurate detection of boundary elements of subjects isdifficult, accurate determination of a colony region is made difficultaccording as a large amount of noise is detected or, in contrast,regions which should have been detected as boundary elements of subjectsare overlooked in extraction of boundary elements of subjects. That is,in extraction of boundary elements of subjects, the following problemsare raised: (1) not only boundary elements of cells, but also boundaryelements of intracellular organelles are detected; (2) overlooking ofboundary elements of cells occurs; (3) three-dimensional mutualoverlapping of, cells complicates boundary elements of the cells.

Therefore, in the cell-image analyzing apparatus of this embodiment, inorder to solve these problems, the colony region determining means isconfigured to use, in accordance with a choice by a user, any of thefirst mode to the fourth mode (Steps S33 ₁-S33 ₄) as a processing modefor determining whether each of cells satisfies Condition (2) as a unitcomponent of a cell collective in a possible colony region in theprocess of colony region determination (Step S33). Explanations are madeon the processing modes optimal for the situations (i)-(iv) below,respectively:

(i) recognition of individual cells is easy;

(ii) small amount of boundary elements of subject is extracted;

(iii) moderate amount of boundary elements of subjects is extracted;

(iv) large amount of boundary elements of subjects is extracted.

Processing Mode for Individually Determining Whether Each of CellsSatisfies Condition First Mode (Step S33 ₁)

The first mode is a processing mode preferable in the situation wherethe cell image is sufficiently clear and is free from three-dimensionaloverlapping of cells, to facilitate recognition of individual cells (thesituation of (i) above). In the first mode, the colony regiondetermining means 1 c determines the regions delimited by boundaryelements of subjects as individual cells, and detects regions of thosecells respectively satisfying Condition (2).

Processing Mode for Individually Determining Whether Each of CellsSatisfies Condition (2) Second Mode (Step S33 ₂)

FIGS. 6A-6D are explanatory diagrams that show one example of thecell-image analysis procedure according to the second mode in thecell-image analyzing apparatus of this embodiment, where FIG. 6A sh theoriginal cell state (cell image), FIG. 6B shows the state of extractingboundary elements of subjects from the cell image of FIG. 6A, FIG. 6Cshows the state of detecting a non-colony cell region, upon setting anon-colony cell model region in the image with the extracted boundaryelements of FIG. 6B, and FIG. 6D shows the state of delimiting regionsof individual cells other than the non-colony cell, upon setting acircle which is a largest circle encircling no boundary element of thesubjects with a radius smaller than the non-colony cell model region, inthe image with the detected non-colony cell region of FIG. 6C.

The second mode is a processing mode preferable in the situation where asmall amount of boundary elements of subjects are extracted and onlyboundaries of individual cells are detected as boundary elements ofsubjects (the situation, of (ii) above). We here suppose, as aparticular situation where the second mode is applied, anexperimentation system in which a few elements are detected as boundaryelements of subjects depending on conditions regarding various factorssuch as boundary elements of intracellular organelles and noise. Also,we suppose that the original cell state in this experimentation systemis as illustrated in FIG. 6A, that boundary elements of subjects areextracted as shown in FIG. 6B, and that the region presented with theboundary elements of subjects shown in FIG. 6B is determined as apossible colony region.

In this case, the colony region determining means 1 c sets, as anon-colony cell model region, in the possible colony region, a circle,an ellipse, a square or other figure with a certain size, which isdetermined in reference to the minimum size of a cell in a non-colonyregion as a criterion. Then the colony region determining means 1 cdetects a region where a non-colony cell exists, upon making thenon-colony cell model region scan (shift the position) in the possiblecolony region. To be specific, if there is a region where the non-colonycell model region is allowed to be set without encircling unda elementof subjects, this region is detected as a region containing a non-colonycell (non-colony cell region).

If it is difficult, to delimit a region in concern in the possiblecolony region by boundary elements of subjects (for example, in the casewhere a simple closed region cannot be formed by boundary elements ofsubjects) the colony region determining means 1 c further sets a largestcircle that has a radius smaller than the non-colony cell model regionand that encircles no boundary element, as shown in FIG. 6D, forexample. By repeating this step of setting a circle inside the possiblecolony region, the colony region determining means is delimits regionsof individual cells other than the non-colony cell.

Processing Mode for Individually Determining Whether Each of CellsSatisfies Condition (2) Third Mode (Step S33 ₃)

FIGS. 7A-7B are explanatory diagrams that show one example of thecell-image analysis procedure according to the third mode in thecell-image analyzing apparatus of this embodiment, where FIG. 7A showsthe state of evaluating intricacy of boundary elements of subjects uponsetting evaluation regions having a uniform shape and size in thepossible colony region, and FIG. 7B shows a series of regions evaluatedas high-density regions regarding boundary elements of subjects.

The third mode is a processing mode preferable in the situation where amoderate amount of boundary elements of subjects are extracted anddelimitation of individual cell regions, if by the second mode, would beaffected by noise of boundary elements of subjects or so (the situationof (iii) above). In the third mode, as shown in FIG. 7A for example, thecolony region determining means 1 c sets evaluation regions having auniform shape and size (in this example, circular regions) in thepossible colony region, digitizes the amount of boundary elements ofsubjects encircled in the evaluation regions in terms of number ofpixels, and gives the detected values as the intricacy. Then, the colonyregion determining means 1 c determines whether each of the evaluationregions is a colony region or a non-colony region in accordance with thedetected intricacy. For example, if the detected intricacy is greaterthan a criterion value preliminarily set for evaluation, the colonyregion determining means 1 c evaluates the inside of the circular regionas a high-density region containing clustered cells forming a colony,and determines a series of regions evaluated, as high-density regions asshown in FIG. 7B as a colony region.

Processing Mode for Individually Determining Whether Each of CellsSatisfies Condition (2): Fourth Mode (Step S33 ₄)

FIGS. 8A-8C are explanatory diagrams that show one example of thecell-image analysis procedure according to the fourth mode in thecell-image analyzing apparatus of this embodiment, where FIG. 8A showsthe basic method of predicting a region occupied by a single cell in thepossible colony region by using boundary elements of a subject, FIG. 8Bschematically shows the state of predicting a region occupied by asingle cell, in the possible colony region using boundary elements ofsubjects in the situation where the boundary elements of subjects existnot only on the circumference of the cell but also inside the cell, andFIG. 8C schematically shows the state of predicting a region occupied bya single cell in the possible colony region using boundary elements ofsubjects in the situation here a part of the boundary elements aremissing.

The fourth mode is a processing mode preferable in the situation where alarge amount of boundary elements of subjects are extracted and analysisof the cell image is difficult by the third mode because of much denserboundary elements of subjects (the situation of (iv) above). In thefourth mode, the colony region determining means 1 c grows a markedregion from an arbitrary origin on the boundary elements of subjects inthe possible colony region by repeating the process of incorporatingadjacent points into the marked region until the marked region containsa preset “average number of boundary pixels”, and predicts that themarked region as containing “average number of boundary pixels” is aregion occupied by a single cell inside the possible colony region.Here, “average number of boundary pixels” is an average value ofboundary pixels (number of pixels) for a single cell.

If a region occupied by a single cell is predicted via the processing ofthe fourth mode, as shown in FIG. 8A for example, a cell having acircular shape occupies a large area and a cell having a complicatedcontour occupies a small area. According to the processing of the fourthmode, as shown in FIG. 8B for example, even in the situation whereboundary elements of subjects exist not only on the circumference of thecell but also inside the cell, a region occupied by the single cell inthe possible colony region is predictable using the boundary elements ofsubjects. Further, in the situation where apart of the boundary elementsis missing (broken) a region occupied by a single cell in the possiblecolony region is predictable using the boundary elements of subjectsupon changing the value the “average number of boundary pixels” inaccordance with the situation.

FIG. 9 is an explanatory diagram that schematically shows the process inwhich, according to the fourth mode, the colony region determining means1 c determines individual cells in a possible colony region thatcontains a plurality of clustered cells. First, the colony regiondetermining means 1 c determines a “single-cell region” starting at anarbitrary origin on the boundary elements of subjects in the possiblecolony region by the fourth mode. Then, the colony region determiningmeans 1 c determines another single-cell region in the similar manner bythe fourth mode, at a position adjacent to this single-cell region asfirst determined. By repeating this processing one after another foradjacent positions, individual cell regions in the possible colonyregion are determined.

In a colony having a complicated structure, boundary elements of eachcell do not necessarily corresponds to the boundary of the cell.Therefore, the cell-image analyzing apparatus of this embodiment isconfigured so that the average value of boundary elements of subjectsper cell is preliminarily set as the “average number of boundary pixels”for processing of the fourth mode by the colony region determining means1 c. This configuration allows individual cell regions having theaverage number of boundary pixels to be determined.

FIGS. 10A and 10B are explanatory diagrams that schematically show cellregions determined by the fourth mode in a possible colony region thatcontains clustered cells, where FIG. 10A schematically shows the statewhere cells are clustered, and FIG. 10B shows regions of individualcells determined by the fourth mode. In FIG. 10B, the square framesrepresent the individual cell regions. Here, a single-cell region isschematically presented as a square. As shown in FIG. 10, individualsingle-cell regions determined by the “average number of boundarypixels” are narrowed as the cells are more densely clustered to formfurther intricate cell boundaries.

A non-colony cell has a simple shape, and a non-colony cell regiondetermined by the fourth mode is different from a cell in a colonyregion in size and morphologic characteristic such as circularity.Therefore, in the fourth mode, the colony region determining means 1 cis configured to classify the individual regions in a possible colonyregion by morphologic characteristic. This technique also makes itpossible to distinguish between a cell in a colony region and a cell ina non-colony region. For example, in a region having intricate boundaryelements such as a colony, single-cell regions determined there areaveraged to have a shape approximate to a circle, as shown in FIG. 11A.On the other hand, a mature cell has a small amount of boundary elementsand is relatively large, with its single-cell region as determined beingapproximate to an ellipse, as shown in FIG. 11B. Therefore, in thefourth mode, it is preferred that the colony region determining means 1c distinguishes whether each of single-cell regions belongs to a colonyregion or a non-colony region in accordance with circularity (parametertaking a smallest value as the shape is a circle, and a larger value asit deviates from the circle).

Processing Corresponding to Condition (3)

Regarding the individual cell regions that have been determined inaccordance with one of the processing modes excluding the third mode(i.e. the first triode, the second mode and the fourth mode) forindividually determining whether cells in concern satisfy Condition (2),the colony region determining means 1 c determines their respectivesize, and estimates the number of cells contained in each of thepossible colony regions using the respective size of the cell regions asdetermined. Then the colony region determining means 1 c determines aregion that contains a number of cells, as estimated, greater than thepredetermined number as a colony region.

Result Output Step (Step S4)

In the analysis result output step (Step S4), the analysis output means1 d outputs parameters, such as the size and the estimated number ofcells, of regions determined via the cell-image analysis step (Step S3)on a display monitor or a printer not shown.

The cell-image analyzing apparatus of the present invention is useful infields where analysis of a collective of clustered cells (cell colony)using an image of cells as cultured is required.

1. A cell-image analyzing apparatus provided with a computer, foranalyzing a cell collective that forms a colony using cell image;wherein the cell-image apparatus comprises an image analysis softwarethat makes the computer function as: a boundary element extracting meansfor extracting boundary elements of subjects such as cells,intracellular organelles or dust upon analyzing the cell image; apossible colony region determining means for determining, as a possiblecolony region, a region surrounded by boundary elements of subjects asextracted via the boundary element extracting means and having a sizegreater than a first criterion value; and a colony region determiningmeans for determining, in the possible colony region determined via thepossible colony region determining means, a region that contains morethan a predetermined number of clustered regions each being surroundedby boundary elements of subject and having a size smaller than a secondcriterion value, as a colony region.
 2. A cell-image analyzing apparatusaccording to claim 1, wherein the boundary element extracting meansextracts, as boundary elements of subjects, pixel portions whereluminance greatly changes, and wherein the colony region determiningmeans has a function of digitizing intricacy of boundary elements ofsubjects and of dividing the possible colony region into a colony regionand a non-colony region in accordance with the intricacy of boundaryelements as digitized.
 3. A cell-image analyzing apparatus according toclaim 2, wherein the colony region determining means sets, in thepossible colony region determined via the possible colony regiondetermining means, evaluation regions having a uniform shape and size,detects values of boundary elements of subjects encircled in theevaluation regions in terms of number of pixels, and gives the detectedvalues as the intricacy.
 4. A cell-image analyzing apparatus accordingto claim 1, wherein the colony region determining means has a functionof setting as a non-colony cell model region, in the possible colonyregion, a circle, an ellipse, a square or other figure with a certainsize, which is determined in reference to a minimum size of a celloutside a colony as a criterion, and of detecting, upon shifting aposition of the non-colony cell model region in the possible colonyregion, a region where the non-colony cell model region is allowed to beset without encircling any boundary element of subjects, as a region ofanon-colony cell.
 5. A cell-image analyzing apparatus according to claim4, wherein the colony region determining means further has a function ofdelimiting regions of individual cells other than the non-colony cell bysetting a largest circle that has a radius smaller than the non-colonycell model region and that encircles no boundary element and repeatingthis step of setting a circle inside the possible colony region.
 6. Acell-image analyzing apparatus according to claim 1, wherein the colonyregion determining means has a function of predicting that, upon growinga marked region from an arbitrary origin on the boundary elements ofsubjects in the possible colony region by repeating a process ofincorporating adjacent points into the marked region until the markedregion contains a preset average number of boundary pixels for a singlecell, the marked region as containing the average number of boundarypixels is a region occupied by a single cell inside the possible colonyregion.
 7. A cell-image analyzing apparatus according to claim 6,wherein the colony region determining means further has a function ofdistinguishing, in the possible colony region, whether each regionpredicted as occupied by a single cell belongs to a colony region or anon-colony region based on a morphologic characteristic.
 8. A cell-imageanalyzing apparatus according to claim 7, wherein the morphologiccharacteristic is circularity.